Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen (LN2). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in 50 μl droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop (47.1±6.9%) than that of French mini straw (15.9±12.5%). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) (84.5±14.2%) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2∼3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above LN2 for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at 50℃ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions’ groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

본 실험은 국화의 바이로이드 제거에 이용되는 초저온처리 시 국화 품종 'White ND'을 적합한 처리조건을 확립하기 위해 초저온처리의 단계별 요인을 실험하였다. 그 결과 생장점의 크기는 1 mm(엽원기 2~3매 포함)에서 높은 생존율과 신초 재생율을 나타내었고, vitirification 처리시 PVS3가 효과적이었으며, 처리 시간은 60분 처리 하였을 때 높은 생존율 및 정 상 신초 재생율을 보였다. 또한 vitrification을 위한 전처리 조건은 sucrose 농도를 88 mM 24시간, sucrose 0.3 M 16시간, sucrose 0.5 M 6시간, sucrose 0.7 M 3시간으로 처리하는 것이 초저온 처리 후 생존율 및 신초 재생율을 높이는데 효과적이었으며, 재생된 정상 식물체는 모본과 비교하여 ploidy level이 동일한 것으로 보아 식물체의 유전적 변이가 일어나지 않았다.

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in 38.5℃, 5% CO2 incubator. For freezing, Day 7∼9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to —7℃, and the straw was seeded during 8 minutes-holding time, and was cooled to —35℃ at the cooling rate of 0.3℃/min, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method (56.86 ± 26.53%) were slightly higher than those by the slow-rate freezing method (55.07 ± 26.43%) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were 72.65 ± 18.3% and 79.06 ± 17.8%, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were 66.22 ± 18.8% and 45.76 ± 12.8%, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst (99.7 ± 12.4) compared to the post-thaw blastocyst (94.8 ± 15.1). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups (74.7 ± 14.6, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different (0.0 ± 0.0 vs. 1.9 ± 3.1, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group (5.4 ± 4.4) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

파이로그린공정의 염폐기물처리과정에서 발생되는 주요 산화물 형태의 폐기물에는 희토류폐기물이 있으며 주요 구성 핵종은 Y, La, Ce, Pr, Nd, Sm, Eu, Gd 등 8종이다. 최종적인 희토류폐기물의 형태는 산화물 형태로 발생된다. 본 연구에서는 붕규산 유리계 내에서 희토류 산화물의 유리화 타당성을 평가 하기 위하여 6종의 유리조성을 개발하였다. 희토류 8핵종 혼합에 대한 solubility는 1,200℃에서 25wt% 미만, 1,300℃에서 30wt% 미만 waste loading으로 온도 상승에 따라 증가하는 것으로 나타났으며 liquidus temperature는 균질한 유리가 형성된 20wt% waste loading에서 950℃ 이하로 평가되었다. 희토류 산화물의 유리매질 내 solubility 이상에서는 희토류-oxide-silicate 결정이 생성된 유리세라믹을 이차상으로 형성하였으며 20~25wt% waste loading의 표면균질성이 양호한 유리는 용융온도 1,200~1,300℃ 범위에서 점도 100 poise 이하, 전기전도도 1 S/cm 이상으로 유도가열식 저온용융로설비에서의 운전 용이성이 매우 양호한 것으로 평가되었다. 개발된 유리조성에 대한 기타 물리·화학적 특성 평가를 위한 실험들이 향후 수행될 예정이다.

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ( vs. , ) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.